Endocrine cells develop within pancreatic bud-like structures derived from mouse ES cells differentiated in response to BMP4 and retinoic acid.

We have examined factors affecting the in vitro differentiation of Pdx1(GFP/w) ESCs to pancreatic endocrine cells. Inclusion of Bone Morphogenetic Protein 4 (BMP4) during the first four days of differentiation followed by a 24-hour pulse of retinoic acid (RA) induced the formation of GFP(+) embryoid bodies (EBs). GFP expression was restricted to E-cadherin(+) tubes and GFP bright (GFP(br)) buds, reminiscent of GFP(+) early foregut endoderm and GFP(br) pancreatic buds observed in Pdx1(GFP/w) embryos. These organoid structures developed without further addition of exogenous factors between days 5 and 12, suggesting that day 5 EBs contained a template for the subsequent phase of development. EBs treated with nicotinamide after day 12 of differentiation expressed markers of endocrine and exocrine differentiation, but only in cells within the GFP(br) buds. Analysis of Pdx1(GFP/w) ESCs modified by targeting a dsRed1 gene to the Ins1 locus (Pdx1(GFP/w)Ins1(RFP/w) ESCs) provided corroborating evidence that insulin positive cells arose from GFP(br) buds, mirroring the temporal relationship between pancreatic bud development and the formation of endocrine cells in the developing embryo. The readily detectable co-expression of GFP and RFP in grafts derived from transplanted EBs demonstrated the utility of Pdx1(GFP/w)Ins1(RFP/w) ESCs for investigating pancreatic differentiation in vitro and in vivo.